Genome-wide identification and expression analysis of the SAUR gene family in foxtail millet (Setaria italica L.)

Background Auxin performs important functions in plant growth and development processes, as well as abiotic stress. Small auxin-up RNA (SAUR) is the largest gene family of auxin-responsive factors. However, the knowledge of the SAUR gene family in foxtail millet is largely obscure. Results In the current study, 72 SiSAUR genes were identified and renamed according to their chromosomal distribution in the foxtail millet genome. These SiSAUR genes were unevenly distributed on nine chromosomes and were classified into three groups through phylogenetic tree analysis. Most of the SiSAUR members from the same group showed similar gene structure and motif composition characteristics. Analysis of cis-acting elements showed that many hormone and stress response elements were identified in the promoter region of SiSAURs. Gene replication analysis revealed that many SiSAUR genes were derived from gene duplication events. We also found that the expression of 10 SiSAURs was induced by abiotic stress and exogenous hormones, which indicated that SiSAUR genes may participated in complex physiological processes. Conclusions Overall, these results will be valuable for further studies on the biological role of SAUR genes in foxtail development and response to stress conditions and may shed light on the improvement of the genetic breeding of foxtail millet. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04055-8.

Lots of SAUR genes have been shown to play an important role in plant growth, development, and stress response in Arabidopsis thaliana. Some AtSAUR genes are involved in auxin mediated cell expansion with their unique expression pattern. For example, Arabidopsis AtSAUR36 [21], AtSAUR41 [22], AtSAUR19 [23], and AtSAUR63 [24], positively regulate cell expansion and promote hypocotyl growth with high expression in hypocotyls, cotyledons, petioles, and flowers. Recent studies have shown that AtSAUR41 is inducible by abscisic acid to regulate salt tolerance [22,25]. Although the function of the SAUR gene family has been extensively reported in Arabidopsis thaliana, it is rarely reported in monocotyledonous rice. There are 58 SAUR gene members in rice [14], but only three members have been cloned: OsSAUR39, OsSAUR45, and OsSAUR33. Compared with the wild type, the transgenic plants overexpressing OsSAUR39 had lower auxin content, reduced auxin polar transport, and showed a significant decrease in tiller and panicle number [26]. OsSAUR45 affects plant growth by inhibiting the expression of OsYUCCA and OsPIN genes and affecting auxin synthesis and transport [27]. Disruption of OsSAUR33 significantly reduced seed vigor and germination rate, but increased soluble sugar content in early germination and mature seeds [28]. However, a comprehensive investigation of SAUR family genes has not been conducted and their special role in auxin signaling is still unknown in foxtail millet.
Foxtail millet (Setaria italic L.) is an important minor crop due to its strong tolerance to drought and barren stress. For its small genome (~ 515 Mb) and short life cycle, foxtail millet has gradually developed into an ideal C4 model plant. Although genome sequencing has been completed in 2013 [29], the functional genomics of foxtail millet is still developing slowly. What we have known and understood in SAUR gene family is too little to comprehensively understand the genetic basis of SAUR genes. Also, the SAUR gene family has not been systematically identified and analyzed in foxtail millet. To better understand the function and evolution of the SAURs in plants, we analyzed the SAUR gene family in foxtail millet. A total of 72 SiSAUR genes were identified here and they were divided into three groups. In addition, comprehensive analysis, including gene structure, conserved motifs, cis-acting elements, gene replication, synteny analyses, expression analysis, and subcellular localization of these genes were all performed.

Identification, characterization, and phylogenetic analysis of SAUR genes in foxtail millet
In our study, the HMM model of SAUR conserved domain was used to verify all possible SAUR members in the S.italica genome. The identified members of the SiSAUR gene family were renamed as SiSAUR1 to SiSAUR72 (Table S1). Among the 72 SiSAUR proteins, SiSAUR26 was the smallest with 75 amino acids (aa), while the largest was SiSAUR13 with 373 aa. The molecular masses and isoelectronic point (pI) of the proteins ranged from 8.21 kDa to 39.49 kDa and from 4.9 (SiSAUR4) to 11.61 (SiSAUR13), with a mean of 8.27, respectively. Seventeen SiSAURs had a pI of less than 7, indicating that they were acidic proteins, while the pI of the rest of the proteins were all higher than 7, indicating that they were basic proteins. The instability index of SiSAUR proteins ranges from 26.57 (SiSAUR3) to 80.62 (SiSAUR10). Almost 90% (65/72) of the SiSAUR proteins had an instability index of more than 40, suggesting that SiSAUR proteins were extremely unstable, which was consistent with the report in [3,14,16]. The aliphatic index of SiSAUR proteins ranges from 53.19 (SiSAUR32) to 104.15 (SiSAUR4), with a mean of 78.55. Based on the average grand of hydropathicity, SiSAUR10, SiSAUR32, SiSAUR43, SiSAUR70, and SiSAUR71 were classified as hydrophilic proteins, while others belong to amphiphilic proteins (Table S1).
The prediction of the secondary structure of these SAUR proteins in millet showed that α-helix, extended strand, β-turn, and random curl were found in each member of the family (Table S2). Among them, α-helix and random curl, accounting for 20% ~ 60% of the total secondary structure, were the major constituent elements of the secondary structure of the gene family that favors the formation of the conformation of a particular protein structure. Extended strand accounted for 5% ~ 27%, β-turn accounted for the least, only at around 6%. Subcellular localization results suggested that 37 SiSAUR proteins were predicted to be in the mitochondria, 21 were predicted to be in the cytoplasm, and 14 were predicted to be in the nucleus. All the 72 SiSAUR family members obtained the conservative domain auxin-inducible (Table S2).
To further investigate the phylogenetic relationship of SAUR proteins, phylogenetic tree was constructed using the protein sequences of the 72 identified SiSAURs, 79AtSAURs [3], and 58 OsSAURs [14] ( Fig. 1, Table S1 and S3). The phylogenetic tree was divided into three main phylogenetic clades, namely Group I, Group II, and Group III. Among these members, the Group III branch was the largest group containing 163 members, Group II contained 32 members, while the Group I branch contained only 14 members ( Fig. 1 and Fig. 2A).

Conserved motifs, gene structures and cis-acting elements analysis of SiSAUR genes
The conserved motifs and distribution of exonintron structures were analyzed to explore the structural diversity of SiSAUR genes (Fig. 2). A total of 15 conserved motifs (motif 1 to motif 15) were identified (Fig. 2B, Table S4). Except for SiSAUR4, all of the other SiSAUR genes contained a motif 1. Interestingly, SiSAUR4 did not contain any motif. Motif  and motif 5 were widely distributed in the SiSAUR family. Interestingly, the motif distribution was different in the three groups of the SiSAUR gene family. Both groups contain motifs 1, 4, and 5, motifs 7 and 8 appear in Group I, motifs 6 and 13 appear in Group II, while motifs 2-3, 9-12, and 14-15 appear in Group III. Although there were differences in motif types between groups, members of the same group such as SiSAUR28 and SiSAUR29, SiSAUR2 and SiSAUR14, SiSAUR46 and SiSAUR57 tend to exhibit similar motif patterns ( Fig. 2A, B), indicating functional similarity between them. The comparison of the number of the exon-intron structures revealed that the 72 SiSAUR genes had different numbers of exons, varying from 1 to 3 (Fig. 2C, Table S1). Most of them (59, ~ 81.9%) contained no intron; 11 SiSAUR genes contained one  (Fig. 2C). Exon-intron structural analysis showed that most members of the SiSAUR gene family have no intron structures, which was consistent with the results in rice [14] and other studies [8,9].
The cis-acting elements in the promoter regions (the upstream 2000 bp from the initiation codon) of 72 SiSAUR genes were further investigated. A total of 46 cis-regulatory elements were identified (Fig. 3, Table  S5), which were mainly divided into three categories: Fig. 3 Cis-elements prediction in the 2000 bp region upstream from the start codon of SiSAURs hormone response elements, stress response elements, and plant growth and development elements. There are five hormone-responsive elements in the SiSAUR gene family of the foxtail millet, which cover most plant hormones, including abscisic acid (ABA), auxin (IAA), gibberellin (GA), methyl jasmonate (MeJA), and salicylic acid (SA) (Fig. 4A). The abscisic acid responsiveness elements (ABRE elements, 64 members), light responsiveness elements (G-box element, 69 members) and MeJA-responsiveness elements (CGTCA-motif and TGACG-motif elements, 65 and 63 members) in the promoter region were identified in almost all the SiSAUR genes. Most of the SiSAUR gene promoter region contained three to four hormone response elements. SiSAUR1, SiSAUR18, SiSAUR21, SiSAUR26, SiSAUR28, SiSAUR38, and SiSAUR72 contained all the five hormone-responsive elements. The MeJA response element had the largest number among the five hormone-responsive elements, followed by the ABA response element. The auxin response element was ranked third. Literatures reported that MeJA and ABA respond to stress [30], so the stress response and plant growth and development related elements were further studied.
As shown in Fig. 4B, SiSAUR2, SiSAUR30, SiSAUR32, and SiSAUR65 contained the largest number of elements, including endosperm expression (GCN4_motif ), low temperature response (LRT), MYB binding site (MBS), seed specific regulation (RY-element), stress responsiveness (TC-rich repeats), and zein metabolism regulation elements (O 2 -site). SiSAUR3, SiSAUR26, SiSAUR27, and SiSAUR44 did not contain any stress elements or plant growth and development elements. Also, the number of genes containing MYB binding site (MBS) elements was the highest, followed by zein metabolism regulation (O 2 -site) and low temperature response (LRT) elements. These results indicated that the SiSAUR genes may be involved in stress response. In addition, some cis-acting elements may regulate the expression of different tissues (seed, root, and endosperm) during development (Table  S5). These results suggested that SiSAUR genes could not only participate in the process of plant growth and development but also respond to various abiotic stresses.

Chromosome locations and gene duplication analysis
The number and location of SiSAUR genes on the chromosomes were also investigated ( Fig. 5A, Table S1). The physical positions of these SiSAUR genes on the chromosomes were visualized using the MapGene2Chrom website. The 72 SiSAUR genes are distributed on nine chromosomes unevenly. Chr2 contained the largest number of SiSAUR genes (22 genes, ~ 30.5%), followed by Chr6 (10, ~ 13.8%); Chr8 contained the fewest SiSAUR genes (3 genes, ~ 4.16%). Chr3 and Chr5 contained the same number of SiSAUR genes (six each, ~ 8.3%). Chr1, Chr7, Chr4, and Chr9 contained nine (~ 12.5%), seven (~ 9.7%), five (~ 6.9%), and four (~ 5.5%) SiSAUR genes, respectively. The SiSAUR genes in three groups also showed an uneven distribution. The genes in Group I were found on Chr2, Chr3, Chr6 and Chr9, whereas the genes in Group III were distributed across all nine chromosomes. There are more Group III genes on Chr2 (Fig. 5B). Also, some SiSAUR genes within the same group tend to cluster together on the chromosome. For example, SiSAUR22 and SiSAUR23, and SiSAUR13 and SiSAUR14, which belong to the Group III and Group II, respectively, are tightly linked on chromosomes 6 (SiSAUR22/23) and 7 (SiSAUR13/14) (Fig. 5). Gene duplication events, which generally include tandem repeats and segmental repeats, are always important for the expansion of gene families [31,32]. Previous literature reported that the inclusion of two or more genes in a 200 kb region was defined as a tandem duplication event [33]. In this study, we found 33 tandem duplication events involving 46 SiSAUR genes on all the chromosomes except chromosome 9 (Fig. 5A). SiSAUR16, SiSAUR19, SiSAUR23, and SiSAUR40 each involved in two tandem repeat events (SiSAUR16 and SiSAUR15/SiSAUR17; SiSAUR19 and SiSAUR18/SiSAUR20; SiSAUR23 and SiSAUR22/SiSAUR24; SiSAUR40 and SiSAUR39/SiSAUR41). Interestingly, 18 SiSAUR genes formed 17 tandem duplication events on chromosome 2. All the SiSAUR genes involving in the tandem repeat events tend to belong to the same subgroup (Table S1). In addition, nine pairs of segmentally duplicated genes were found in the SiSAUR family (Fig. 6, Table S6). The nine segmental duplications were unevenly distributed into eight linkage groups (LG). The distribution of SiSAUR genes was largest in LG I and LG IV (each 4). LG II and LG V, and LG VI had three and two SiSAUR genes, whereas LG III, LG VII, and LG IX had only one SiSAUR gene (Table S6). These findings showed that some SiSAUR genes might be due to some events in gene replication, which might have been the primary driving factor for SiSAUR gene development and evolution.

Synteny analyses of SiSAUR genes
To investigate syntenic relationship of SiSAUR genes, the collinearity analysis was conducted between SiSAUR genes and five other representative species (three monocotyledons: Oryza sativa, Zea mays, and Sorghum bicolor; two diocotyledons: Arabidopsis thaliana and Solanum lycopersicum) (Fig. 7, Table S7). We found that totals of 41 SiSAUR genes showed collinear relationships with Arabidopsis (4), Solanum lycopersicum (8), Sorghum bicolor (30), Oryza sativa (18), and Zea mays (35). The collinearity map revealed that SiSAUR genes showed the highest collinearity with maize, followed by Sorghum bicolor, rice, Solanum lycopersicum and Arabidopsis. Further analysis of these collinear genes revealed that some SiSAUR genes were found to exist in more than one collinear gene pair in five species, such as SiSAUR42 with AT1G72430.1/ Os09t0437100-00/ Zm00001eb100000_ T001/ OQU89560/ Solyc07g042490.1.1, which indicated that these collinear genes may have existed prior to the ancestral separation and divergence. What's more, six SiSAUR genes (SiSAUR11, 14, 21, 31, 43 and 71) are present in both monocotyledons and diocotyledons (Table  S7). Among these six genes, three genes (SiSAUR21, 31, and 43) were also included in the genes with segmental duplications (Fig. 5A, Table S6). And some SiSAUR genes showed collinearity only with maize (eight genes: SiSAUR3, 17,27,35,36,38,40, and 63) and Sorghum bicolor (five genes: SiSAUR13, 22, 23, 28, and 37) (C4 plant). In addition, the number of genes with collinearity to the monocots was much higher than that with collinearity to the dicots. The result that foxtail millet exhibited the highest collinearity with maize and Sorghum bicolor, suggesting that these C4 plants may have a closer genetic relationship.

Organ expression pattern analysis of SiSAURs
To determine the expression of SiSAURs in various tissues of foxtail millet, the transcription data of SiSAURs in different tissues of Yugu1 were analyzed (Fig. 8). As shown in Fig. 8, the expression level of SiSAURs in different stages and tissues is mainly divided into three groups. The genes in group A were highly expressed in the whole development stage of foxtail millet. SiSAUR71, SiSAUR72, SiSAUR35, and SiSAUR3 were highly expressed in root, stem, leaf, panicle, and seed, indicating that these four genes may play an important role during the growth and development of foxtail millet. SiSAUR34 was specifically highly expressed in developing panicles, which may be related to panicle development. Group B genes have a relatively low expression level in the vegetative growth stage. Some group C gene expressions were not detected in any stage or tissue, such as SiSAUR27, SiSAUR28, and so on. The different expression patterns suggested that the functions of SiSAURs had been differentiated in long-term evolution.

SiSAUR gene expression was induced by multiple abiotic stresses
According to the analysis of cis-elements in the promoter region of SiSAURs, SiSAURs might be involved in the abiotic stresses and hormone response processes. To further verify this hypothesis, the expression patterns of 10 SiSAUR genes belonging to Group I (SiSAUR4, SiSAUR18, SiSAUR20), Group II (SiSAUR27, SiSAUR28, SiSAUR34) and Group III (SiSAUR48, SiSAUR50, SiSAUR54, SiSAUR61), which were treated with 20% PEG-6000, 150 mM NaCl, ABA, SA, and GA, respectively, were detected by RT-qPCR (Fig. 9). These 10 SiSAUR genes had different responses to the various abiotic stresses and phytohormone treatments. They showed various expression patterns over time under different treatments. For example, after 2 h of ABA treatment, the expression of SiSAUR4 and SiSAUR28 genes was significantly inhibited, then recovered with the increase of treatment time. The expression of the SiSAUR34 gene was significantly induced, and SiSAUR18 and SiSAUR27 showed a similar induced expression pattern, which was inhibited after 2 h of ABA treatment, then recovered rapidly, and after 24 h of treatment, the gene expression was inhibited again (Fig. 9A). Under GA treatment, the expression patterns of these 10 genes showed a trend of down-regulation first, then up-regulation and then down-regulation, similar to the expression pattern of SiSAUR18 and SiSAUR27 under ABA treatment (Fig. 9B). Under SA treatment, the expression level of SiSAUR4 was significantly induced. SiSAUR28, SiSAUR34, SiSAUR27, SiSAUR54, and SiSAUR61 showed different degrees of expression change, which was inhibited for 2 h after treatment, and then significantly up-regulated (Fig. 9C). When treated with 150 mM IAA, these 10 genes were all up-regulated, and the gene expression reached its highest level after 10 h of treatment (Fig. 9D). The obvious upregulation of the expression level under IAA treatment may be due to the fact that the promoter region of these SAUR genes is rich in a large number of auxin cis-acting elements ( Fig. 3 and Fig. 4A). In PEG and salt stress conditions, these genes were induced to different degrees ( Fig. 9E and F). The expression levels of SiSAUR4 and SiSAUR18 were significantly up-regulated under 150 mM NaCl and 20% PEG-6000 treatments, indicating that these two genes may be involved in drought stress. These results suggested that SiSAUR genes play various regulatory roles in abiotic stress and phytohormone treatment.

Subcellular localization of SiSAUR proteins
According to the phylogenetic tree result, five genes, which came from different group (Group I: SiSAUR38; Group II: SiSAUR29, SiSAUR41; Group III: SiSAUR12, SiSAUR24), were picked to detect the location of these SiSAUR proteins (Fig. 10). The coding region with the stop codon removed was cloned into the PS1300 vector carrying the green fluorescent protein (GFP) gene, and then transformed into rice protoplast cell. The results showed that the GFP fluorescence signal of SiSAUR29-GFP was widely present in the cell membrane, nucleus, and cytoplasm, while SiSAUR41-GFP signal was found in the cell membrane, nuclear membrane, and cytoplasm (Fig. 10A). The GFP fluorescence signal of SiSAUR12, SiSAUR24 and SiSAUR38 were just existed in nucleus (Fig. 10B).

SiSAUR gene structure and its evolutionary analysis
In this study, the SiSAUR gene family was thoroughly investigated, and over all 72 SiSAUR genes were discovered (Table S1). The SAUR protein has a length range from 75 to 373 amino acids, which may explain the sequence variability and complexity of SAUR. Among the 72 SAUR genes identified, 59 genes lacked intron, which also appeared in rice, sorghum, and other crops [14,15,17,18]. Thirteen genes had introns, which might be involved in some biological processes like mRNA output and alternative splicing [34]. The imbalanced distribution of SAUR genes across each chromosome could be a sign that the genetic diversity has been there throughout the course of evolution. These SAUR genes were classified into three groups based on their evolutionary links with the known Arabidopsis and rice SAUR genes, indicating the indispensable role of these SAUR genes in the development and evolution of S. italica (Fig. 1, Table  S1 and S3). According to the evolutionary tree, group III had a larger number of S. italica members (n = 51, 70.8%), similar to that in Arabidopsis (n = 68, 86.1%) and rice (n = 51, 70.8%), suggesting that these SAUR genes in group III may have undergone a more rapid expansion during the long process of evolution under the influence of monocotyledons. Additionally, each group's gene members not only share similar gene structure, motif structure, and protein length, but also have the same outcomes in predicting subcellular localization (Table S1 and S2). These findings imply that the SAUR gene family members, particularly those belonging to the same subfamily, may play more stable roles in foxtail millet growth. The similarities and variations in the gene structure, motif, and domain of SiSAURs may be linked to the extended evolutionary history as well as gene replication of foxtail millet. Segmented replication and tandem repetition are the main driving forces in the development of gene family extension and genomic evolution [31]. We discovered 33 events of tandem repeat comparising 46 SiSAUR genes (Fig. 5A) and nine segment duplication pairs (Fig. 6, Table S6). Therefore, SiSAUR gene tandem duplication might have a stronger impact on the SAUR family's amplification and evolution in foxtail millet. As expected, all these genes were mainly within the same group, similar to A.thaliana [8,9], and rice [14]. Collinear analysis also displayed that a majority of the SAUR homologous gene pairs existed in foxtail millet, Zea mays and Sorghum bicolor (Fig. 7, Table S7). Based on these findings, we hypothesized that whole-genome replication may be the key factor encouraging the proliferation of the SAUR genes.

Expression patterns of SiSAURs under abiotic stresses
Here, qRT-PCR analysis was conducted to reveal the expression characteristics of SiSAUR genes under multiple phytohormone and abiotic treatments. Among these ten SiSAUR genes selected, the expression of SiSAUR48 was obviously induced under various treatments (Fig. 9). Significant intron loss was clearly evident in most SiSAUR members (Table S1). These could result in family expansion and give rise to new roles and functions. In plants, genes having few or no introns are normally expressed at low levels. Compact-structured genes, on the other hand, may facilitate and ease the rapid expression of genes in response to abiotic stress [35]. For example, the expression of SiSAUR4 increases rapidly under phytohormone, PEG, and NaCl stress treatments, and may be in response to these abiotic stresses (Fig. 9).
It is well known that the promoter regulates the temporal and spatial expression of a gene, and the cisacting elements in the promoter region are crucial for gene function. Therefore, the cis-acting elements in the promoter region of SiSAUR genes were analyzed and large numbers of stress-responsive cis-acting elements, including hormone response elements, stress response elements, and plant growth and development elements, were found (Fig. 3, Table S5). ABA has been shown in studies to play a very crucial role in regulating stress responses in plants [36]. ABA response elements (ABREs) were identified in the cis-acting elements of the SiSAUR gene family. Also, qRT-PCR analysis revealed that many SiSAURs positively responded to stress in the short time of salt and drought treatments ( Fig. 9E and F). Meanwhile, the qRT-PCR results of the SiSAUR family members indicated that they could be induced by various abiotic stresses, which were well consistent with previous reported results [37,38]. These results suggest that the SiSAUR gene family may regulate the drought and salt stress responses, especially for genes SiSAUR4, SiSAUR18, SiSAUR27, SiSAUR48, and SiSAUR54, which need further experimental verification.

Conclusions
A total of 72 SAUR genes have been discovered in the genome of foxtail millet. The characteristics, chromosomal distribution, phylogenetic relationships, conserved motifs, gene structure, cis-acting elements, gene replication, and synteny analyses of the 72 SiSAUR genes were characterized. These SiSAUR genes have been distributed unevenly across nine chromosomes and organized into three groups. The structure analysis of SiSAUR genes also revealed that the majority of them have no introns, suggesting that they are conserved. In addition, we discovered that both tandem and segment duplications are significant contributors to the expansion of the SiSAUR gene family, with tandem duplication possibly having a greater impact. In addition, the expression patterns of 10 SiSAUR genes were analyzed in response to different abiotic stresses, including PEG and NaCl stress, and different phytohormone treatments, including IAA, ABA, GA, and SA. The expression levels of these ten SiSAUR genes were all upregulated after IAA treatment, and these genes were sensitive to abiotic stresses. These results provide evidence of the relationship between SiSAUR genes and abiotic stresses, and they may be a valuable resource for traditional foxtail millet breeding.

Identification of the SiSAUR family genes
The complete genome of Foxtail millet was retrieved from the Ensemble Plants Genomes website (https:// plants. ensem bl. org/ index. html). Foxtail millet SAUR sequences were downloaded through BLAST methods. First, was used to validate the candidate SAUR proteins and then we downloaded the hidden Markov model (HMM) file corresponding to the SAUR domain (PF02519) from the database of the Pfam protein family (http:// pfam. xfam. org/). The SAUR protein sequences were obtained using HMMER3.0 with a cutoff of 0.01 (http:// plants. ensem bl. org/ hmmer/ index. html) from the foxtail millet genomic database. Finally, the NCBI Conserved Domain Database (https:// www. ncbi. nlm. nih. gov/ Struc ture/ cdd/ wrpsb. cgi) and SMART database (http:// smart. embl-heide lberg. de/) were used to confirm whether the candidate protein sequences contained the SAUR core domain. Moreover, the basic characteristics of the trihelix proteins encoded by the SAUR genes of S. italica were determined using ExPasy (https:// web. expasy. org/ protp aram/). SOPMA (https:// npsa-prabi. ibcp. fr/ cgi-bin/ npsa_ autom at. pl? page= npsa_ sopma. html) and Psort (https:// www. gensc ript. com/ psort. html) were used to predict the secondary structure and subcellular localization of SAUR proteins, respectively.

Phylogenetics, gene structures and conserved motif analysis
The phylogenetic tree was generated using the Neighbor-Joining (NJ) method with 1000 bootstrap replications in MEGA11 [39]. The Evolview online tool was used to annotate and visualize the resulting tree [40]. Also, the predicted coding sequences were compared to their full-length sequences through the online program Gene Structure Display Server (http:// gsds. gao-lab. org/ index. php), which was used to determine the exon-intron structure of SiSAUR genes [41]. The Multiple EM for Motif Elicitation (https:// meme-suite. org/ index. html) online program was used to discover the conserved motif in the SiSAUR gene proteins [42]. These parameters were carried out to search for: the number of motifs is 20, while the motif width ranges from 6 to 50 residues. Finally, the outcomes of the gene structure as well as conserved motif analysis were visualized using TBtools software [43].

Cis-regulatory element analysis of SiSAUR genes
The complete genome of foxtail millet was retrieved from the Ensemble Plants Genomes website (https:// plants. ensem bl. org/ index. html). TBtools software was used to obtain the 2000 bp sequence upstream of the initiation codon of SiSAUR genes. Then PlantCARE software was then used to predict the 2000 bp upstream cis-acting elements in the SiSAUR genes (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/). Finally, the results were visualized using TBtools software.

Chromosomal distribution and gene duplication
The physical locations of SiSAUR genes were determined using the genome annotation files, which were downloaded from the Ensemble Plants Genomes website. The SiSAUR gene positions on the chromosome were visualized through MG2C software (http:// mg2c. iask. in/ mg2c_ v2.1/). The TBtools software was used to scan the collinearity of SiSAUR genes and analyze gene-duplication events. TBtools was also used to analyze the collinear relationship between SAUR genes of foxtail millet, maize, rice, Arabidopsis, Solanum lycopersicum, and Sorghum bicolor by default parameters, and the collinear gene pairs between millet and maize, rice, Arabidopsis, Solanum lycopersicum, and Sorghum bicolor were obtained.

Plant materials, growth conditions and treatments
In our study, the foxtail millet accession (Yugu 1) was used and obtained from Li Junxia of Henan Academy of Agricultural Sciences. The seeds of Yugu1 were soaked overnight and pregerminated on moistened filters in a plant growth chamber under the condition of 16 h light/8 h dark (28 °C/24 °C) with 65% humidity for 3 days. Then the seedlings were transferred into a hydroponic box with a modified Hoagland nutrient solution. When the third leaf was unfolded (approximately 4 weeks), the seedlings were treated with 20% PEG-6000, 150 mM NaCl, 150 mM IAA, 150 mM SA,150 mM ABA and 150 mM GA. Samples were taken at 0, 2, 4, 8, 10, 12 and 24 h, respectively. All of the samples were frozen immediately in liquid nitrogen and stored at -80 °C for further assays.

RNA extraction and qRT-PCR analysis
Total RNA was extracted from shoots of each treatment using RNAiso Plus (Takara, Japan), and cDNA was generated using an M-MLV reverse transcriptase (Takara, Japan). qRT-PCR was performed on an ABI 7500 Realtime PCR system (Applied Bio-Systems). SiActin7 was used as an internal reference. Each experiment was performed with five biological samples, and each sample was assayed with three technical replications. The relevant gene primers are listed in Table S8. The experimental data ware analyzed using the 2 −(△△CT) method [44].